应用纳米磁珠实时荧光PCR检测非洲木薯花叶病毒

Detection of African Cassava Mosaic Virus by Real-time Fluorescent PCR Combining with Magnetic Nanoparticles

建立非洲木薯花叶病毒(African cassava mosaic virus,ACMV)灵敏快速的纳米磁珠实时荧光PCR检测技术,防止其传入中国。采用TaqMan探针结合纳米磁珠(magnetic nanoparticles,MNPs)技术,以外壳蛋白基因为目标基因,通过特异性及灵敏度试验建立MNP荧光PCR检测方法。成功建立了非洲木薯花叶病毒(ACMV)的MNP荧光PCR检测方法;该方法检测阈值约为13pg DNA,比普通PCR及ELISA方法灵敏度高25倍;该方法具有良好的特异性,能够有效区分双生病毒属的ACMV、棉花曲叶病毒(CLCuV)、番茄黄化曲叶病毒(TYLCV)及番茄严重曲叶病毒(ToSLCV)。MNP荧光PCR方法能够快速灵敏的检测茎叶样品中的ACMV,无需任何PCR后处理,交叉污染风险小,适于口岸检验检疫。

The aim of establishing a sensitive and rapid real-time fluorescent PCR combining with magnetic nanoparticles method to detect African cassava mosaic virus（ACMV） was to prevent the ACMV from introducing into China.By using TaqMan probe combining with magnetic nanoparticles technique and coat protein gene as the target gene,the MNP fluorescent PCR detection method was established after specificity and sensitivity experiments.The MNP fluorescent PCR detection technique was successfully developed.The sensitivity of the method was 13 pg of DNA,which was 25 times higher than that of the conventional PCR and ELISA method.The method had good specificity and could effectively distinguish four geminiviruses,ACMV,Tomato yellow leaf curl virus（TYLCV）,Cotton leaf curl virus（CLCuV） and Tomato severe leaf curl virus（ToSLCV）.The MNP fluorescent PCR method was rapid and accurate for ACMV detection of stem and leaf samples and could reduce the risk of cross-contamination without any PCR post-processing,which could be applied to the field of port inspection and quarantine.